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Glucose utilization in muscle during uremia; in vitro study
CLINICA CHIMICA ACTA
GLUCOSE
UTILIZATION
R. DZtJRIK
AND
IlIrd
EVA
IN MUSCLE DURING
UREMIA:
IN V1TRO
STUDY
VALOVICOVA
Medical Clinic. Come&us
(Received
I37
University Medical School, Bratislava (Czechoslovakia)
April 8th, 1970)
SUMMARY I. Rat hemidiaphragms incubated in uremic sera utilize less glucose than those incubated in control sera. Uremic sera do not inhibit the utilization of fructose. 2. The concentrations of free glucose, glucose 6-phosphate and fructose 6-phosphate in diaphragms incudated in uremic sera rise, the concentration of fructose 1,6diphosphate falls, indicating the crossover point at the level of phosphofructokinase. 3. Uremic sera decrease the concentrations of pyruvate, citrate and a-ketoglutarate in diaphragms, but do not influence the concentrations of adenosine tri-, di-, or monophosphate. It is concluded that uremic sera inhibit primarily the activity of phosphofructokinase in diaphragms. The significance of citrate or adenosine phosphates in evoking this inhibition was excluded.
INTRODUCTION
Decreased glucose utilization in muscle participates in the abnormal carbohydrate metabolism in uremia : Westervelt 1,2 found, that insulin stimulates glucose uptake in the forearms of uremic patients insufficiently, when compared with the forearms of control subjects. Moreover, sera obtained from the uremic patients inhibit glucose utilization in rat diaphragms 3. The utilization of lactate and a-ketoglutarate, as well as oxygen consumption are not inhibited. It was concluded, that uremic sera inhibit either transmembrane transport of glucose or glycolysis in diaphragms3. The present paper deals with localization of the limiting step which causes the inhibition of glucose utilization in rat diaphragms. MATERIALS
AND METHODS
Blood samples were obtained from 12 patients with chronic uremia. The primary cause of uraemia was chronic glomerulonephritis (7 patients) and pyelonephritis (5 patients). All the patients had blood urea concentration over 150 mg/roo ml and abnormal glucose tolerance test. None of the patients had primary diabetes or was ever dialyzed. Specimens of control blood were taken from normal volunteers. C&n. Chim. Acta, 30
(1970) 137-142
DZURIK, VALOVICOVA
138
Blood was collected in the morning after an overnight fast, allowed to stand for 30 min at room temperature before centrifuging and the serum, after separation, was centrifuged again. The serum was then diluted with an equal volume of Kinger bicarbonate, containing 5 mM glucose. Only in the experiment for the examination of fructose utilization, Ringer bicarbonate contained 20 mM fructose instead of glucose. Male Wistar rats, 120-150 g weight, last fed 20 h before the experiment, were decapitated and their diaphragms removed, immediately chilled in isotonic saline and hemidiaphragms put in uremic or control sera alternatively. They were incubated at 37’ in a Dubnoff apparatus in an atmosphere of 95 “/
concentrafreezed by
by the hexokinase,
glucose 6-phosphate dehydrogenaseB method. Lactate’, pyruvates, citrates, a-ketoglutaratelO, ATPI’, ADP and AMP12, glucose 6-phosphate13, fructose 1,6-diphosphate14 enzymatically. For the determination of glucose, lactate, pyruvate, cr-ketoglutarate, ATP, ADP and AMP, Boehringer commercial kits were used. Glucose 6-phosphate dehydrogenase, hexose isomerase, aldolase, triose isomerase and glycerol I-phosphate dehydrogenase and citrate lyase were Boehringer products. Glycogen was determined by the anthrone method16 after two precipitations with ethanol. Fructose was determined by the method of KulkaIB. Analyses were performed in duplicate. Percentage of dry weight in diaphragms incubated in control sera was 21.5 & 0.26; in those incubated in uremic sera it was 22.0 + 0.23. Because the uremic sera did not influence the amount of water content, all the results were calculated on wet tissue weight. KESULTS Glucose utilization Rat diaphragms incubated in the uremic sera for I h utilize significantly less glucose than those incubated in control sera (Table I). Uremic sera stimulate glycogen utilization only insignificantly. The amount of glucose gained from the stimulation of glycogenolysis represents only 3% of the amount of glucose which was not utilized by diaphragms because of the inhibitory action of the uremic sera. Thus, from this point of view glycogenolysis can be neglected. TABLE
I
UTILIZATION OF GLUCOSE,GL.YCOGEN ANDFRUCTOSE Utilization
Control seva pmoles/g
Glucose Glycogen * * Fructose (IO mM)
fresh
Uremic
43.4 + 1.3’3 (9)* I.55 f 1.21 (9) 26.1 * 1.46 (6)
17.2 & 1.76 (9) 2.36 + 9.89 (9) 27.3 ?C I.54 (6)
* Mean value + S.E.M. (number of investigated * * Glycogen is expressed in ,umoles of glucose. C&Z. Chim.
Acta,
30
(1970)
137-142
sera
P
weight in I h
sex-a).
< 0.001
GLUCOSE UTILIZATION
IN UREMIA
I39
In the next experiment the time-course of glucose utilization was studied, glucose uptake and lactate production being measured after 20,40 and 60 min (Fig. I). The inhibition of glucose utilization and lactate production were marked during the whole period of time. Thus, the r-h incubation represents the whole time-course and it was used in most of the following experiments. 40-PM glucose/g wet
20-
weight
*--*Uremic
sera
0-o
sera
Control
f -1 30.
Fig. I. Time-course of glucose utilization and
Localization
of the ilzhibitory
lactate
production.
efect
From the point of view of regulations at the cellular level two possibilities exist: (I) Uremic sera stimulate breakdown of some noncarbohydrate metabolites. Glucose uptake decreases secondarily. For instance, increased supplementation of the citric acid cycle by several intermediates originating from fatty acids or amino acids would cause the accumulation of citric acid cycle intermediates. The increased concentration of citrate would inhibit the activity of phosphofructokinase17-‘8 and secondarily the utilization of glucose. (2) Uremic sera inhibit primarily glucose utilization. The concentrations of citric acid cycle intermediates decreases and stimulate the breakdown of other substrates. To decide, which of these two possibilities holds true, the concentrations of pyruvate, citrate and a-ketoglutarate were measured in diaphragms. Uremic sera decreased the concentrations of all the three acids (Table II). Thus, uremic sera inhibit primarily glucose utilization and the site of inhibition takes place before the synthesis of pyruvate. Normal fructose utilization by diaphragms incubated in the uremic sera (Table I) excludes even the possibility of inhibition at the triose phosphate level. Next, it was studied, whether uremic sera inhibit the transmembrane transport or the first steps of glycolysis. The significance of transmembrane transport was tested by measuring free glucose concentration in diaphragms. If no free glucose is present intracellulary, the ratio muscle glucose/medium glucose varies about 0.20 (ref. 20). Clin. Chim. Ada,
30 (1970)
137-142
DZORIK,
140 TABLE
VALOVICOV.4
II
CONCENTRATION
Substrate
OF SOME
INTERMEDIATES
IN
Control sera nmoles/g
Pyruvate Citrate a-Ketoglutarate ATP
P
Uremic seva
wet weight
4oo * I97 i 75 zt 1010 *
31.9 (8) 3.6 (12) I.4 (6) (II) 57 612 & 30 (II) 380 & 26 (II)
ADP AMP
DIAPHRAGM
299 & 25.0 (8) 151 zk 3.6 (12) 61 rt 4.4 (6) 1116
&
g6
654 f 28 4oo zk 25
< 0.05 < 0.001 < 0.02
(II)
(II) (II)
-
At higher ratios free glucose is present intracellularly. Tissue glucose/medium glucose ratio varied in diaphragms incubated in control sera around 0.36 and uremic sera still increased this ratio (Table III). This excludes the limiting effect of uremic sera at the level of transmembrane transport. Thus, glucose utilization is limited at the level of glycolysis, namely at the level of hexose phosphates. To decide, where the inhibition takes place, the concentrations of glucose and hexose phosphates were measured (Fig. 2). The crossover point was localized at the level of phosphofructokinase. It is possible, therefore, to conclude that uremic sera decrease glucose utilization by inhibiting the activity of phosphofructokinase. TABLE
III
TISSUE GLUCOSE/MEDIUM GLUCOSE RATIO Incubation
(min)
20
60
Control
SeYa
0.361 & 0.0041 (7) 0.354 =k o.o1*8 (7)
Uremic zeta
P
0.415 5 0.0160 (7) 0.396 i 0.0043 (7)
< 0.05 < 0.05
Crossover
/point
Fig. 2. The effect of uremic sera on the concentration phragm.
of glucose and hexose phosphates
in dia-
The n?,echanisnzof inhibition Modulation of phosphofructokinase activity is rather complex17-19. From the physiological point of view two regulatory mechanism are of special interest : (I) The inhibitory action of citrate. However, it was found (Table II) that the citrate concenClin. Chim. Acta,
30 (1970)
137-142
GLUCOSE UTILIZATION
141
IN UREMIA
tration of diaphragms in uremic sera decreased. (2) The inhibitory action of ATP and stimulatory effect of ADP and AMP. Nevertheless, the concentration of these nucleotides does not change in such a manner (Table III) that it would explain the inhibition of phosphofructokinase activity by this mechanism. Inhibitor, present in the uremic sera cannot inhibit the activity of phosphofructokinase by influencing these modulators. It is highly probable, that it inhibits phosphofructokinase activity directly. However, at least partial purification of inhibitor is necessary before making any conclusions about the mechanism of its effects. DISCUSSION
The inhibition of phosphofructokinase activity by the uremic sera as a primary cause of decreased glucose utilization in uremia fits well with the observations of Westerveltlpa, who examined the response of intact human forearm to brachial intraarterial insulin infusion and measured glucose, lactate, potassium and inorganic phosphorus fluxes. On the basis of his results he proposed the inhibition of phosphorylation as a cause of decreased glucose utilization found in uremic patients. The inhibition of phosphofructokinase activity fits well also with the finding that though glucose tolerance in uremia decreases, fructose tolerance does not change21. During the last years two hypotheses were postulated to elucidate the mechanism of glucose utilization : (I) Serum contains an insulin antagonist and the decrease of glucose utilization reflects the decreased effectiveness of insulin22. (2) Decreased glucose utilization reflects some changes in metabolic activity of muscle, independent on insulin antagonism21z3. Though insulin modulates the activity of phosphofructokinase2*, our results fit better with the hypothesis of independent inhibition of glucose utilization. Nevertheless, it is possible, that an inhibitor (or inhibitors) present in uremic sera decrea.ses glucose utilization directly and antagonizes insulin activity. In any case, the abnormality is not caused only by intracellular changes, but is evoked by modulator (or modulators ?) present in uremic sera: Hemidiaphragm from the same rat incubated in uremic serum utilizes less glucose than the one incubated in control serum. Now, it seems necessary to define better this modulator. ACKNOWLEDGEMENTS
The authors wish to express their gratitude to Mrs. A. Matygkova and A. Obornikova for their excellent technical assistance and to Dr. Zankl from Boehringer Biochemical Department for the gift of hexose isomerase, triose isomerase and glycerol I-P dehydrogenase. REFERENCES I B. F. WESTERVELT,
2 3 4 5 6
Jr., Am. J. Clin. N&r., 21 (1968)423. B. F. WESTERVELT, Jr., J. Lab. Clin. Med., 74 (1969) 79. R. DZCJRIK AND B. KRAJEI-LAZARY, Experientia, 23(1967) 798. A. WOLLENBERGER, 0. RISTAU AND G. SCHOFFA, Pftiigers Arch. Ges. Physiol., 270 (1960) 399. A. S. G. HUGGET AND D. A. NIXON, Biochem. J., 66 (1957) 12P. M. V. SLEIN, in H. U. BERGMEYER, Methods of Enzymatic Analysis, Academic Press, New York,
1963, p. 117. 7 F. WR~BLEWSKI
AND J. S. LADUE,
Proc. Sot. Exptl.
Biol. Med.,
go (1955) 210.
Cl&. Chim. Acta,
30 (1970) 137-142
DZtiRIK,
142
VALOVICOVA
8 T. BOCHER, R. CZOK, W. LAMPRECHT AND E. LATZKO, in H. U. BERGMEYER, Methods of Enzymatic Analysis, Academic Press, New York, 1963, p. 253. g H. MOELLERING AND W. GRUBER, Anal. Biochem., 17 (1966) 369. IO H. U. BERGMEYER AND E. BERNT, in H. U. BERGMEYER, Methods of Enzymatic Analysis, Academic Press, New York, 1963, p. 324. II H. ADAM, ibid., p. 539. 12 H. ADAM, ibid., p. 537. 13 H. J. HOHORST, ibid., p. 134. I4 T. B~CHER AND H. J. HOHORST, ibid., p. 246. 15 D. L. MORRIS, Science, 107 (1948) 254. 16 R. G. KULKA, Biochem. /., 63 (1956) 542. 17 A. H. UNDERWOOD AND E. A. NEWSHOLME, Biochem. J., 95 (1965) 868. 18 D. GARFINKEL, J. Biol. Chem., 241 (1966) 286. Ig T. E. MANSOUR, Phavmacol. Rev., 18 (1966) 173. 20 G. SCHONFELD AND D. KIPNIS, Diabetes, 17 (1968) 422. 21 R. G. LUKE, A. J, DINWOODIE, A. L. LINTON ANI) A. C. KENNEDY, J. Lab. CL&. Med., 64 (1964)
731. zz J, D. BRIGGS, K. D. BUCHANAN, R. G. LUKE AND M. T. MCKIDDIE, Lancet, i (1967) 462. 23 M. B. DAVIDSON, E. G. LOWRIE AND C. L. HAMPERS, Metabolism, 18 (1969) 387. 24 P. OZAND AND H. T. NARAHARA, J. Biol. Chem., 239 (1964) 3146. Clin. Chim.
Acta, 30 (1970) 137-142
GLUCOSE
UTILIZATION
R. DZtJRIK
AND
IlIrd
EVA
IN MUSCLE DURING
UREMIA:
IN V1TRO
STUDY
VALOVICOVA
Medical Clinic. Come&us
(Received
I37
University Medical School, Bratislava (Czechoslovakia)
April 8th, 1970)
SUMMARY I. Rat hemidiaphragms incubated in uremic sera utilize less glucose than those incubated in control sera. Uremic sera do not inhibit the utilization of fructose. 2. The concentrations of free glucose, glucose 6-phosphate and fructose 6-phosphate in diaphragms incudated in uremic sera rise, the concentration of fructose 1,6diphosphate falls, indicating the crossover point at the level of phosphofructokinase. 3. Uremic sera decrease the concentrations of pyruvate, citrate and a-ketoglutarate in diaphragms, but do not influence the concentrations of adenosine tri-, di-, or monophosphate. It is concluded that uremic sera inhibit primarily the activity of phosphofructokinase in diaphragms. The significance of citrate or adenosine phosphates in evoking this inhibition was excluded.
INTRODUCTION
Decreased glucose utilization in muscle participates in the abnormal carbohydrate metabolism in uremia : Westervelt 1,2 found, that insulin stimulates glucose uptake in the forearms of uremic patients insufficiently, when compared with the forearms of control subjects. Moreover, sera obtained from the uremic patients inhibit glucose utilization in rat diaphragms 3. The utilization of lactate and a-ketoglutarate, as well as oxygen consumption are not inhibited. It was concluded, that uremic sera inhibit either transmembrane transport of glucose or glycolysis in diaphragms3. The present paper deals with localization of the limiting step which causes the inhibition of glucose utilization in rat diaphragms. MATERIALS
AND METHODS
Blood samples were obtained from 12 patients with chronic uremia. The primary cause of uraemia was chronic glomerulonephritis (7 patients) and pyelonephritis (5 patients). All the patients had blood urea concentration over 150 mg/roo ml and abnormal glucose tolerance test. None of the patients had primary diabetes or was ever dialyzed. Specimens of control blood were taken from normal volunteers. C&n. Chim. Acta, 30
(1970) 137-142
DZURIK, VALOVICOVA
138
Blood was collected in the morning after an overnight fast, allowed to stand for 30 min at room temperature before centrifuging and the serum, after separation, was centrifuged again. The serum was then diluted with an equal volume of Kinger bicarbonate, containing 5 mM glucose. Only in the experiment for the examination of fructose utilization, Ringer bicarbonate contained 20 mM fructose instead of glucose. Male Wistar rats, 120-150 g weight, last fed 20 h before the experiment, were decapitated and their diaphragms removed, immediately chilled in isotonic saline and hemidiaphragms put in uremic or control sera alternatively. They were incubated at 37’ in a Dubnoff apparatus in an atmosphere of 95 “/
concentrafreezed by
by the hexokinase,
glucose 6-phosphate dehydrogenaseB method. Lactate’, pyruvates, citrates, a-ketoglutaratelO, ATPI’, ADP and AMP12, glucose 6-phosphate13, fructose 1,6-diphosphate14 enzymatically. For the determination of glucose, lactate, pyruvate, cr-ketoglutarate, ATP, ADP and AMP, Boehringer commercial kits were used. Glucose 6-phosphate dehydrogenase, hexose isomerase, aldolase, triose isomerase and glycerol I-phosphate dehydrogenase and citrate lyase were Boehringer products. Glycogen was determined by the anthrone method16 after two precipitations with ethanol. Fructose was determined by the method of KulkaIB. Analyses were performed in duplicate. Percentage of dry weight in diaphragms incubated in control sera was 21.5 & 0.26; in those incubated in uremic sera it was 22.0 + 0.23. Because the uremic sera did not influence the amount of water content, all the results were calculated on wet tissue weight. KESULTS Glucose utilization Rat diaphragms incubated in the uremic sera for I h utilize significantly less glucose than those incubated in control sera (Table I). Uremic sera stimulate glycogen utilization only insignificantly. The amount of glucose gained from the stimulation of glycogenolysis represents only 3% of the amount of glucose which was not utilized by diaphragms because of the inhibitory action of the uremic sera. Thus, from this point of view glycogenolysis can be neglected. TABLE
I
UTILIZATION OF GLUCOSE,GL.YCOGEN ANDFRUCTOSE Utilization
Control seva pmoles/g
Glucose Glycogen * * Fructose (IO mM)
fresh
Uremic
43.4 + 1.3’3 (9)* I.55 f 1.21 (9) 26.1 * 1.46 (6)
17.2 & 1.76 (9) 2.36 + 9.89 (9) 27.3 ?C I.54 (6)
* Mean value + S.E.M. (number of investigated * * Glycogen is expressed in ,umoles of glucose. C&Z. Chim.
Acta,
30
(1970)
137-142
sera
P
weight in I h
sex-a).
< 0.001
GLUCOSE UTILIZATION
IN UREMIA
I39
In the next experiment the time-course of glucose utilization was studied, glucose uptake and lactate production being measured after 20,40 and 60 min (Fig. I). The inhibition of glucose utilization and lactate production were marked during the whole period of time. Thus, the r-h incubation represents the whole time-course and it was used in most of the following experiments. 40-PM glucose/g wet
20-
weight
*--*Uremic
sera
0-o
sera
Control
f -1 30.
Fig. I. Time-course of glucose utilization and
Localization
of the ilzhibitory
lactate
production.
efect
From the point of view of regulations at the cellular level two possibilities exist: (I) Uremic sera stimulate breakdown of some noncarbohydrate metabolites. Glucose uptake decreases secondarily. For instance, increased supplementation of the citric acid cycle by several intermediates originating from fatty acids or amino acids would cause the accumulation of citric acid cycle intermediates. The increased concentration of citrate would inhibit the activity of phosphofructokinase17-‘8 and secondarily the utilization of glucose. (2) Uremic sera inhibit primarily glucose utilization. The concentrations of citric acid cycle intermediates decreases and stimulate the breakdown of other substrates. To decide, which of these two possibilities holds true, the concentrations of pyruvate, citrate and a-ketoglutarate were measured in diaphragms. Uremic sera decreased the concentrations of all the three acids (Table II). Thus, uremic sera inhibit primarily glucose utilization and the site of inhibition takes place before the synthesis of pyruvate. Normal fructose utilization by diaphragms incubated in the uremic sera (Table I) excludes even the possibility of inhibition at the triose phosphate level. Next, it was studied, whether uremic sera inhibit the transmembrane transport or the first steps of glycolysis. The significance of transmembrane transport was tested by measuring free glucose concentration in diaphragms. If no free glucose is present intracellulary, the ratio muscle glucose/medium glucose varies about 0.20 (ref. 20). Clin. Chim. Ada,
30 (1970)
137-142
DZORIK,
140 TABLE
VALOVICOV.4
II
CONCENTRATION
Substrate
OF SOME
INTERMEDIATES
IN
Control sera nmoles/g
Pyruvate Citrate a-Ketoglutarate ATP
P
Uremic seva
wet weight
4oo * I97 i 75 zt 1010 *
31.9 (8) 3.6 (12) I.4 (6) (II) 57 612 & 30 (II) 380 & 26 (II)
ADP AMP
DIAPHRAGM
299 & 25.0 (8) 151 zk 3.6 (12) 61 rt 4.4 (6) 1116
&
g6
654 f 28 4oo zk 25
< 0.05 < 0.001 < 0.02
(II)
(II) (II)
-
At higher ratios free glucose is present intracellularly. Tissue glucose/medium glucose ratio varied in diaphragms incubated in control sera around 0.36 and uremic sera still increased this ratio (Table III). This excludes the limiting effect of uremic sera at the level of transmembrane transport. Thus, glucose utilization is limited at the level of glycolysis, namely at the level of hexose phosphates. To decide, where the inhibition takes place, the concentrations of glucose and hexose phosphates were measured (Fig. 2). The crossover point was localized at the level of phosphofructokinase. It is possible, therefore, to conclude that uremic sera decrease glucose utilization by inhibiting the activity of phosphofructokinase. TABLE
III
TISSUE GLUCOSE/MEDIUM GLUCOSE RATIO Incubation
(min)
20
60
Control
SeYa
0.361 & 0.0041 (7) 0.354 =k o.o1*8 (7)
Uremic zeta
P
0.415 5 0.0160 (7) 0.396 i 0.0043 (7)
< 0.05 < 0.05
Crossover
/point
Fig. 2. The effect of uremic sera on the concentration phragm.
of glucose and hexose phosphates
in dia-
The n?,echanisnzof inhibition Modulation of phosphofructokinase activity is rather complex17-19. From the physiological point of view two regulatory mechanism are of special interest : (I) The inhibitory action of citrate. However, it was found (Table II) that the citrate concenClin. Chim. Acta,
30 (1970)
137-142
GLUCOSE UTILIZATION
141
IN UREMIA
tration of diaphragms in uremic sera decreased. (2) The inhibitory action of ATP and stimulatory effect of ADP and AMP. Nevertheless, the concentration of these nucleotides does not change in such a manner (Table III) that it would explain the inhibition of phosphofructokinase activity by this mechanism. Inhibitor, present in the uremic sera cannot inhibit the activity of phosphofructokinase by influencing these modulators. It is highly probable, that it inhibits phosphofructokinase activity directly. However, at least partial purification of inhibitor is necessary before making any conclusions about the mechanism of its effects. DISCUSSION
The inhibition of phosphofructokinase activity by the uremic sera as a primary cause of decreased glucose utilization in uremia fits well with the observations of Westerveltlpa, who examined the response of intact human forearm to brachial intraarterial insulin infusion and measured glucose, lactate, potassium and inorganic phosphorus fluxes. On the basis of his results he proposed the inhibition of phosphorylation as a cause of decreased glucose utilization found in uremic patients. The inhibition of phosphofructokinase activity fits well also with the finding that though glucose tolerance in uremia decreases, fructose tolerance does not change21. During the last years two hypotheses were postulated to elucidate the mechanism of glucose utilization : (I) Serum contains an insulin antagonist and the decrease of glucose utilization reflects the decreased effectiveness of insulin22. (2) Decreased glucose utilization reflects some changes in metabolic activity of muscle, independent on insulin antagonism21z3. Though insulin modulates the activity of phosphofructokinase2*, our results fit better with the hypothesis of independent inhibition of glucose utilization. Nevertheless, it is possible, that an inhibitor (or inhibitors) present in uremic sera decrea.ses glucose utilization directly and antagonizes insulin activity. In any case, the abnormality is not caused only by intracellular changes, but is evoked by modulator (or modulators ?) present in uremic sera: Hemidiaphragm from the same rat incubated in uremic serum utilizes less glucose than the one incubated in control serum. Now, it seems necessary to define better this modulator. ACKNOWLEDGEMENTS
The authors wish to express their gratitude to Mrs. A. Matygkova and A. Obornikova for their excellent technical assistance and to Dr. Zankl from Boehringer Biochemical Department for the gift of hexose isomerase, triose isomerase and glycerol I-P dehydrogenase. REFERENCES I B. F. WESTERVELT,
2 3 4 5 6
Jr., Am. J. Clin. N&r., 21 (1968)423. B. F. WESTERVELT, Jr., J. Lab. Clin. Med., 74 (1969) 79. R. DZCJRIK AND B. KRAJEI-LAZARY, Experientia, 23(1967) 798. A. WOLLENBERGER, 0. RISTAU AND G. SCHOFFA, Pftiigers Arch. Ges. Physiol., 270 (1960) 399. A. S. G. HUGGET AND D. A. NIXON, Biochem. J., 66 (1957) 12P. M. V. SLEIN, in H. U. BERGMEYER, Methods of Enzymatic Analysis, Academic Press, New York,
1963, p. 117. 7 F. WR~BLEWSKI
AND J. S. LADUE,
Proc. Sot. Exptl.
Biol. Med.,
go (1955) 210.
Cl&. Chim. Acta,
30 (1970) 137-142
DZtiRIK,
142
VALOVICOVA
8 T. BOCHER, R. CZOK, W. LAMPRECHT AND E. LATZKO, in H. U. BERGMEYER, Methods of Enzymatic Analysis, Academic Press, New York, 1963, p. 253. g H. MOELLERING AND W. GRUBER, Anal. Biochem., 17 (1966) 369. IO H. U. BERGMEYER AND E. BERNT, in H. U. BERGMEYER, Methods of Enzymatic Analysis, Academic Press, New York, 1963, p. 324. II H. ADAM, ibid., p. 539. 12 H. ADAM, ibid., p. 537. 13 H. J. HOHORST, ibid., p. 134. I4 T. B~CHER AND H. J. HOHORST, ibid., p. 246. 15 D. L. MORRIS, Science, 107 (1948) 254. 16 R. G. KULKA, Biochem. /., 63 (1956) 542. 17 A. H. UNDERWOOD AND E. A. NEWSHOLME, Biochem. J., 95 (1965) 868. 18 D. GARFINKEL, J. Biol. Chem., 241 (1966) 286. Ig T. E. MANSOUR, Phavmacol. Rev., 18 (1966) 173. 20 G. SCHONFELD AND D. KIPNIS, Diabetes, 17 (1968) 422. 21 R. G. LUKE, A. J, DINWOODIE, A. L. LINTON ANI) A. C. KENNEDY, J. Lab. CL&. Med., 64 (1964)
731. zz J, D. BRIGGS, K. D. BUCHANAN, R. G. LUKE AND M. T. MCKIDDIE, Lancet, i (1967) 462. 23 M. B. DAVIDSON, E. G. LOWRIE AND C. L. HAMPERS, Metabolism, 18 (1969) 387. 24 P. OZAND AND H. T. NARAHARA, J. Biol. Chem., 239 (1964) 3146. Clin. Chim.
Acta, 30 (1970) 137-142